Parasites feeding on plants affect a variety of crops, especially those cultivated in tropical and Mediterranean climates, presenting a major agricultural problem causing damage estimated in the range of billions of dollars. Such parasites include parasites whose life cycle comprises at least one stage of consumption of plant cytoplasm, including insects, nematodes, and fungi. One approach for reducing such parasite damage is by generating resistant plants, either by genetic selection of natural resistant varieties or by introducing genetic resistance artificially.
Inhibitory RNA (RNAi) has become an important tool for silencing genes in many eukaryotes. It was discovered in the nematode Caenorhabdtitis elegans (Guo, S. and Kemphues, K. J. 1995. Cell 81:611-620) and was later shown to exist in Trypanosoma brucei, Drosophila, Neurospora and more recently in plants and mammalian cells (Wianny, F. and Zernicka-Goetz, M., 2000. Nat. Cell Biol. 2:70-75). In plants, post-transcriptional gene silencing (PTGS), known as co-suppression is mechanistically related to RNAi (see for example, Napoli, C. Lemiex C and Jorgensen R. 1990. Plant Cell 2:279-289). In C. elegans the phenomenon was demonstrated to be triggered by administration of dsRNA to the whole organism (Tabara, H. Grishok A. and Mello C. C. 1998. Science 282:430-431).
WO 98/53083 describes constructs and methods for enhancing the inhibition of a target gene within an organism, which involve inserting into a gene-silencing vector an inverted repeat sequence for all or part of a polynucleotide region within the vector. The inverted repeat sequence may be a synthetic polynucleotide sequence or comprise a modified natural polynucleotide sequence.
WO 99/32619 discloses a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with a double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequence of the duplex region of the RNA and of a portion of the target gene are identical.
WO 99/49029 relates to a method of modifying gene expression and to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. Recombinant DNA technology is used to post-transcriptionally modify or modulate the expression of a target gene in a cell, tissue, or organ, by introducing multiple copies of a nucleotide sequence which is substantially identical to the nucleic acid sequence of the target gene or to the complementary sequence of the target gene under conditions sufficient for translation of the mRNA of the target gene to be modified.
WO 99/53050 discloses methods and means for reducing the phenotypic expression of a nucleic acid of interest in eukaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or, alternatively, by introducing the RNA molecules themselves. Preferably, the RNA molecules comprise simultaneously both sense and antisense nucleotide sequences. Specifically, the methods are directed towards reducing viral infection, or towards reducing the phenotypic expression of endogenous plant gene.
WO 00/49035 discloses a method for silencing the expression of an endogenous gene in a cell, the method involving overexpressing in the cell a nucleic acid molecule of the endogenous gene, wherein the overexpression of the nucleic acid molecule of the endogenous gene and the antisense molecule in the cell silences the expression of the endogenous gene.
U.S. Pat. No. 6,423,885 discloses methods for reducing the phenotypic expression of a nucleic acid of interest in plant cells, by providing aberrant, preferably unpolyadenylated, target-specific RNA to the nucleus of the host cell.
US Application No. 2002/0169298 discloses a method for producing transgenic cereal plants resistant to Barley Yellow Dwarf Virus, particularly in the presence of co-infecting Cereal Yellow Dwarf Virus, by stably integrating into the cells of the transgenic plant a chimeric gene enabling the transcription of a viral RNA dependent RNA polymerase comprising both sense and antisense RNA, capable of pairing and forming a double stranded RNA molecule or hairpin RNA.
The methods described hitherto are all directed to silencing the expression of a target gene within eukaryotic cells, particularly plant cells. To prevent the development of a parasite in or on a plant, silencing an essential parasite gene, particularly a gene involved with the early stages of the parasite establishment, is desired. Gene silencing employing the method of introducing dsRNA was demonstrated in the nematode C. elegans by direct administration of the dsRNA to the parasite. The dsRNA was administered to the worm by various modes of delivery such as microinjection, feeding on Escherichia coli expressing dsRNA or simply soaking the animals in dsRNA preparations (Fire, A. Xu S. Montgomery M. K. Kostas S. A. Driver S. E. Mello C. C. 1998. Nature 391:806-811; Tabara, H. et al., supra).
WO 04/005485, corresponding to US Patent Application No. 2004/0098761 to Trick et al. relates to compositions and methods for controlling nematode infestation of plants or animals. Specifically, the invention discloses transgenic plants transformed with RNAi targeted to an RNA sequence selected from the group consisting of nematode Major sperm protein (MSP), RNA polymerase II and Chitin synthase. The invention is exemplified by reduction in the number of nematode cysts in transgenic plants expressing RNAi targeted to MSP gene.
Nematode infestation is responsible only for a portion of the damage caused by plant parasites to agricultural crops. Other parasites, including insects and fungi feeding on the plant cytoplasm adversely affect the yield of food crops as well as of ornamental crops. Moreover, the growing demand to employ practices suitable for sustainable agriculture, requires the development of new means for parasite control in order to reduce the use of insecticides and fungicides,
Thus, there is a recognized need for, and it would be highly advantageous to have methods of means for controlling plant parasite insects and fungi. Moreover, efficient control of plant parasites requires means directed to arrest the parasite growth at an early developmental stage, so as to prevent its development in or on the plant.